Steroid Structural Requirements for Stabilizing or Disrupting Lipid Domains

The cause was subsequently shown to be related to blockade of the ultimate reaction in the biosynthesis of cholesterol ( 2 ). Recessive mutations were described of the gene coding for 7-dehydrocholesterol reductase, the enzyme responsible for catalyzing the fi nal reduction of 7-dehydrocholesterol to cholesterol. A variety of deleterious mutations for 7-dehydrocholesterol reductase with distinct geographic distribution have been shown ( 3 ) and traced with a noticeably high frequency around 1% in healthy heterozygote carriers ( 4 ).The accumulation in fetal tissue of the metabolite 7-dehydrocholesterol (7-DHC) and derivative isomer 8-dehydrocholesterol is a specifi c biomarker for antenatal diagnosis of this severe condition ( 5 ) involving the central nervous system ( 6 ), masculinization, and facial and limb defects ( 7,8 ). Indeed the accumulation of precursors is constantly associated with a severe defi cit of the end-product cholesterol, which is also judged deleterious for embryo development. The teratology was originally studied by this laboratory in animal models treated with potent inhibitors of cholesterol biosynthesis ( 9 ). The results of in vivo studies are considered inconclusive with respect to whether the cholesterol defi cit or the accretion of aberrant sterol competing with membrane cholesterol is the underlying mechanism for teratology. The administration of a diet highly enriched in cholesterol to inhibitor-treated pregnant dams was proven to suppress malformations in the offspring ( 10 ). Nevertheless, it cannot be concluded that the defi cit in cholesterol alone is causal because negative Abstract The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fl uorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (L o ) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38°C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20°-50°C. Calculated electron density profi les indicated the location of 7-DHC was more loosely defi ned than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome. -Staneva, G., C. Chachaty, C. ...

Section: Discussionmentioning

confidence: 99%

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

Staneva

Chachaty

Wolf

et al. 2010

“…Anisotropy is high in tightly packed gel state and low in loosely packed Ld state (49)(50)(51). The phase transition is detected as a sigmoidal decrease in DPH anisotropy as temperature is increased, with Tm defi ned as the midpoint of the sigmoidal curve ( 52,53 ). case of PS, the Tm of exchange vesicles was stable after 1 day, both with or without PE.…”

Section: Estimating the Stability And Extent Of Asymmetrymentioning

confidence: 99%

The dependence of lipid asymmetry upon polar headgroup structure

Son

London

2013

membrane surrounding the bacterial cytoplasm, it has been reported that phosphatidylinositol (PI) is preferentially located in the inner leafl et, phosphatidylglycerol (PG) is mainly located in the outer leafl et, and cardiolipin (CL) is distributed over both leafl ets in gram-positive bacteria Micrococcus lysodeikticus ( 5 ). Studies from Bacillus megaterium suggested PE mainly resides in the inner leafl et ( 6 ). In the outer membranes of gram-negative bacteria lipopolysaccharides are predominantly localized in the outer leafl et, while phospholipids are enriched in the inner leafl et ( 7-9 ). Several important functional roles of cellular membrane are closely associated with an asymmetrical lipid distribution. For example, dissipation of membrane lipid asymmetry, resulting in PS externalization on the outer leafl et of membrane, facilitates cell recognition and phagocytosis by macrophages during apoptosis ( 10-18 ). Exposure of PS on the outer cell surface has also been known to be involved in physiologically important phenomena including blood coagulation ( 19,20 ), cell adhesion ( 21-23 ), and myotube formation ( 24 ).To emulate cell membranes more closely, a number of methods have been developed to try to obtain asymmetric lipid bilayers (model membranes) ( 25-45 ). However, methods to form asymmetric lipid vesicles with a wide variety of lipid compositions and highly controlled lipid distribution in each leafl et have been lacking. To tackle this problem, our laboratory has developed methods to prepare asymmetric vesicles using methyl-␤ -cyclodextrin The inner and outer leafl et of many cellular membranes exhibits a difference in lipid composition. This difference is called lipid asymmetry. For example, in eukaryotic cells, the choline-containing lipids sphingomyelin (SM) and phosphatidylcholine (PC) are predominantly located in the outer leafl et of the plasma membrane, while the aminecontaining phospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS) are largely or fully confi ned to the plasma membrane inner leafl et ( 1-4 ). Membrane asymmetry is also observed in prokaryotic cells. Although it is diffi cult to determine lipid distribution in the leafl ets of the

“…The bilayer of SM-containing vesicles undergoes a readily detected phase transition from the ordered gel phase to the liquid-disordered phase as temperature is increased. The Tm can be defi ned as the midpoint of this phase transition ( 23,24 ). It was previously found that Tm values are higher in asymmetric SMo/PCi vesicles than symmetric vesicles of the same composition ( 17 ).…”

Section: Assessment Of Asymmetry In Exchange Vesicles Compared With Smentioning

confidence: 99%

The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure

Son

London

2013

stability of membranes, and membrane shape ( 4-10 ). Therefore, biologically realistic asymmetric vesicles could be widely useful in model membrane studies. However, artifi cial membranes produced in the laboratory usually have a symmetric distribution of lipids in the inner and outer leafl ets of the bilayer. Asymmetric artifi cial membranes have been diffi cult to produce, although some important progress has been made ( 11-16 ). We recently developed a robust method in which methyl-␤ -cyclodextrin (M ␤ CD)-induced lipid exchange is used to prepare model membrane vesicles with lipid asymmetry, and we have applied the method to produce asymmetric small, large, and giant unilamellar vesicles (17)(18)(19).In the present study, we extended the range of this method by using it to construct asymmetric vesicles containing SM in the outer leafl et and various species of PCs in the inner leafl et. PC acyl chain structure was systematically varied, and how this variation affects the ability to form asymmetric membranes was assayed. We found that whether the resulting vesicles were asymmetric [SM outside/ PC inside (SMo/PCi)] depended upon PC acyl chain structure. Despite this limitation, asymmetric vesicles with a wide range of acyl chain structures could be produced. This is an important advance that should allow new applications of asymmetric vesicles. These studies also allowed analysis of the effect of lipid type upon membrane structure and behavior. We investigated the origin of the acyl chain dependence of asymmetry by measuring the transverse diffusion rate of the various PCs used. There was a correlation between asymmetry and transverse diffusion. Asymmetric vesicles could be prepared only when PCs with slow transverse diffusion were used. It was found that the presence of two polyunsaturated acyl chains or overly short acyl chains prevented the maintenance of asymmetry. This Abstract Lipid asymmetry, the difference in inner and outer leafl et lipid composition, is an important feature of biomembranes. By utilizing our recently developed M ␤ CDcatalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was effi ciently introduced into the outer leafl et of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To defi ne the origin of this behavior, transverse diffusion (fl ip-fl op) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesic...